Alphavirus vectors having attentuated virion structural proteins

ABSTRACT

The present invention provides immunogenic compositions and methods that may be used to administer safer (i.e., attenuated) alphavirus vectors (such as alphavirus vectors comprising a VEE virion shell) that retain improved immunogenicity as compared with other attenuated alphaviruses (e.g., the VEE 3014 mutant, described below). In particular embodiments of the invention, the alphavirus vector comprises VEE structural proteins comprising an attenuating mutation in the E1 glycoprotein. In other particular embodiments, the attenuating mutation is in the fusogenic region of the E1 glycoprotein. The present invention enables administration of lower dosages of a safer (i.e., attenuated) virus and, thus, can further reduce manufacturing costs. The present inventors have found that immunogenicity of alphavirus vectors may be influenced by a number of factors including species, site and route of administration.

RELATED APPLICATION INFORMATION

This application claims the benefit of U.S. Provisional Application No.60/390,774, Filed 21 Jun. 2002, the disclosure of which is incorporatedherein by reference in its entirety.

GOVERNMENT SUPPORT

The present invention was made with government support under grantnumbers 5P01 A146023 and 5R01 Al51990 from the National Institutes ofHealth. The United States Government has certain rights to thisinvention.

FIELD OF THE INVENTION

The present invention provides improved immunogenic compositions, inparticular, improved immunogenic compositions comprising attenuatedalphavirus virion shells and methods of administering the same in vitroand in vivo.

BACKGROUND OF THE INVENTION

Venezuelan Equine Encephalitis virus (VEE) is a positive-sense RNA virusresponsible for the mosquito-borne epidemic encephalomyelitis in humansand a wide variety of equids in tropical and sub-tropical areas of theNew World. Initial studies to develop a vaccine against encephalyticdisease lead to the development of an attenuated, live virus vaccine byintroducing a variety of attenuating mutations into the virulentparental genome. As an outgrowth of the studies characterizing thebiological consequences of these attenuating mutations, the use ofreplication-defective virus particles, termed viral replicon particles,has shown great promise as a viral vector delivery system. Replicons areconstructed to carry one or more heterologous antigens in place of someor all of the structural genes. The replicons are introduced into targetcells along with a helper construct(s) that expresses the viralstructural protein(s) not encoded by the replicon or, alternatively, thereplicon is introduced into a packaging cell capable of expressing thestructural proteins. The replicons then express the introducedheterologous antigen(s) at very high levels from the subgenomic mRNA.Subsequent viral progeny are prevented from assembly since the repliconsdo not encode all of the essential viral packaging genes. Studies withthe replicon system have shown great promise as vector systems asdemonstrated by their ability to: (1) target to lymphoid tissue, (2)express high levels of antigen, (3) induce protective humoral, cellularand mucosal immune responses that give protection against challenge, and(4) respond to boost after a primary response (e.g., the boost is notprecluded by pre-existing immunity to the vector itself).

As described above, alphavirus replicon particles have been developedwith attenuating mutations so as to increase the safety of virusadministration. Unfortunately, however, attenuating mutations have beenassociated with a decrease in potency, resulting in the need to deliverlarger doses of particles carrying such attenuating mutations to obtainthe desired immunological response following virus administration.Accordingly, there remains a need in the art for improved alphavirusvaccines that have the features of both safety and efficacy.

SUMMARY OF THE INVENTION

The present invention provides immunogenic compositions and methods thatmay be used to administer safer (i.e., attenuated) alphavirus vectors(such as alphavirus vectors comprising a VEE virion shell) that retainimproved immunogenicity as compared with attenuated alphaviruses (e.g.,the VEE 3014 mutant, described below). In particular embodiments of theinvention, the alphavirus vector comprises VEE structural proteinscomprising an attenuating mutation in the E1 glycoprotein. The presentinvention enables administration of lower dosages of a safer (i.e.,attenuated) virus and, thus, can further reduce manufacturing costs. Thepresent inventors have found that immunogenicity of alphavirus vectorsmay be influenced by a number of factors including species, site androute of administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Primary anti-HA response in mice to HA-VRP immunization. Micewere challenged with HA-VRP-3000 or HA-VRP 3014, and bled after 28 days.ELISA assays were performed as described in Example 1.

FIG. 2. Secondary anti-HA response in mice to HA-VRP immunization. At 28days following primary inoculation, mice were boosted with a secondadministration of HA-VRP-3000 or HA-VRP 3014, and bled 28 days followingbooster administration. ELISA assays were performed as described inExample 1.

FIG. 3. CTL response to HIV Clade C gag in mice primed and boosted withHIV_(gag)-VRP-3000.

FIG. 4. Effect of VRP-replicon coat protein on CTL response in miceprimed and boosted with HIV Clade C gag VRP with wild-type (VRP-3000)and mutant (VRP-3014) coat protein at an effector/target ratio of 25:1.

FIG. 5. Effect of different VRP-replicon coat proteins on immunization.Mice were inoculated with HA-VRP-3000 (wild-type), HA-VRP-3014,HA-VRP-3040, and HA-VRP3042 (mutant) as described in Example 4.

FIG. 6. Effect of mode of administration of HA-VRP on Anti-HA response.Mice were inoculated via footpad, subcutaneous, or intradermalinoculation as described in Example 5, boosted at 28 days, and bled at28 days following booster inoculation.

FIG. 7. Targeting of dendritic cells with GFP-VRP in macaques.GFP-VRP-3000 (wild-type) was administered to rhesus macaques asdescribed in Example 6, and inguinal lymph nodes were harvested 18 hourspost-injection. Fluorescence microscopy was performed as described inExample 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention addresses the need in the art for improvedattenuated alphavirus vectors. The alphavirus vectors of the inventioncomprise attenuated virion shells or coats (e.g., a VEE coat) but retainimproved immunogenicity as compared with other attenuated alphaviruses(e.g., the VEE 3014 mutant, described below). Thus, the presentinvention may enable administration of lower dosages of a safer (i.e.,attenuated) virus and, thus, may further reduce manufacturing costs. Thepresent invention is further based on the finding that theimmunogenicity of the alphavirus may be enhanced by both the site androute of administration.

The terminology used in the description of the invention herein is forthe purpose of describing particular embodiments only and is notintended to be limiting of the invention. As used in the description ofthe invention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

Except as otherwise indicated, standard methods known to those skilledin the art may be used for the construction and use of recombinantnucleotide sequences, vectors, helper constructs, transformed hostcells, selectable markers, alphavirus vectors, viral infection of cells,production of attenuated viruses, and the like. Such techniques areknown to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULARCLONING: A LABORATORY MANUAL 3rd Ed. (Cold Spring Harbor, N.Y., 2001);F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (GreenPublishing Associates, Inc. and John Wiley & Sons, Inc., New York).

I. Definitions.

The term “alphavirus” has its conventional meaning in the art, andincludes Eastern Equine Encephalitis virus (EEE), Venezuelan EquineEncephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus,Western Encephalitis virus (WEE), Sindbis virus, including TR339, SouthAfrican Arbovirus No. 86 (S.A.AR86), Girdwood S.A. virus, Ockelbo virus,Semliki Forest virus, Middelburg virus, Chikungunya virus, O'Nyong-Nyongvirus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyamavirus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroavirus, Babanki virus, Kyzlagach virus, Highlands J virus, Fort Morganvirus, Ndumu virus, Buggy Creek virus, and any other virus classified bythe International Committee on Taxonomy of Viruses (ICTV) as analphavirus.

In particular embodiments of the invention, the alphavirus has a VEEvirion shell. According to this embodiment, the alphavirus may be achimeric alphavirus and have a genomic RNA from another alphavirus.Alternatively, the alphavirus virion comprises a VEE E1 glycoprotein andmay comprise structural proteins (e.g., capsid and/or E2 glycoprotein)from other alphaviruses. In other embodiments, the alphavirus is a VEEvirus having both a VEE coat and genomic RNA.

An “Old World alphavirus” is a virus that is primarily distributedthroughout the Old. World. Alternately stated, an Old World alphavirusis a virus that is primarily distributed throughout Africa, Asia,Australia and New Zealand, or Europe. Exemplary Old World virusesinclude SF group alphaviruses and SIN group alphaviruses. SF groupalphaviruses include Semliki Forest virus, Middelburg virus, Chikungunyavirus, O'Nyong-Nyong virus, Ross River virus, Barmah Forest virus, Getahvirus, Sagiyama virus, Bebaru virus, Mayaro virus, and Una virus. SINgroup alphaviruses include Sindbis virus, South African Arbovirus No.86, Ockelbo virus, Girdwood S.A. virus, Aura virus, Whataroa virus,Babanki virus, and Kyzylagach virus.

The complete genomic sequences, as well as the sequences of the variousstructural and non-structural proteins are known in the art for numerousalphaviruses and include: Sindbis virus genomic sequence (GenBankAccession Nos. J02363, NCBI Accession No. NC_(—)001547), S.A.AR86genomic sequence (GenBank Accession No. U38305), VEE genomic sequence(GenBank Accession No. L04653, NCBI Accession No. NC_(—)001449),Girdwood S.A genomic sequence (GenBank Accession No. U38304), SemlikiForest virus genomic sequence (GenBank Accession No. X04129, NCBIAccession No. NC_(—)003215), and the TR339 genomic sequence (Klimstra etal., (1988) J. Virol. 72:7357; McKnight et al., (1996) J. Virol.70:1981); the disclosures of which are incorporated herein by referencein their entireties.

The phrase “alphavirus structural protein(s)” or “VEE structuralprotein(s)” as used herein refers to one or more of the proteins thatare required to produce a functional alphavirus/VEE virion shell. Thealphavirus/VEE structural proteins include the capsid protein, E1glycoprotein, E2 glycoprotein, E3 protein and 6K protein. As usedherein, the term alphavirus “virion shell” is intended to refer to thealphavirus capsid and E1 and E2 glycoproteins assembled to form anenveloped nucleocapsid-like structure. The E3 and 6K alphavirus proteinsare processed out of the mature virus. As described in more detailbelow, certain attenuating mutations are known to affect thisprocessing. As previously described, the alphavirus capsid proteinassociates with itself and with the RNA genome to form the icosahedralnucleocapsid, which is then surrounded by a lipid envelope covered witha regular array of transmembranal protein spikes, each of which consistsof a heterodimeric complex of the two alphavirus glycoproteins, E1 andE2 (See Paredes et al:, (1993) Proc. Nat. Acad. Sci. USA 90, 9095-99;Paredes et al., (1993) Virology 187, 324-32; Pedersen et al., ( 1974) J.Virol. 14:40).

An alphavirus or VEE “genomic RNA” indicates the alphavirus/VEE RNAtranscript. The wild-type alphavirus genome is a single-stranded,messenger-sense RNA, modified at the 5′-end with a methylated cap, andat the 3′-end with a variable-length poly (A) tract. The viral genome isdivided into two regions: the first encodes the nonstructural orreplicase proteins (nsP1-nsP4) and the second encodes the viralstructural proteins (Strauss and Strauss, Microbiological Rev. (1994)58:491-562). As used herein, the term “genomic RNA” encompassesrecombinant alphavirus genomes (e.g., containing a heterologousnucleotide sequence(s)), viral genomes containing one or moreattenuating mutations, deletions, insertions, and/or otherwise modifiedviral genomes. For example, the “genomic RNA” may be modified to form adouble-promoter molecule or a replicon (each as described below).

A “chimeric” alphavirus as used herein comprises an alphavirus virionshell from one alphavirus and a genomic RNA from another alphavirus. Inembodiments of the invention, the chimeric alphavirus comprises VEEstructural proteins. In other particular embodiments, the alphaviruscomprises the VEE E1 glycoprotein.

An “infectious” alphavirus or VEE particle is one that can introduce thealphavirus/VEE genomic RNA into a permissive cell, typically by viraltransduction. Upon introduction into the target cell, the genomic RNAserves as a template for RNA transcription (i.e., gene expression). The“infectious” alphavirus particle may be “replication-competent” (i.e.,can transcribe and replicate the alphavirus genomic RNA) and“propagation-competent” (i.e., results in a productive infection inwhich new alphavirus particles are produced). In embodiments of theinvention, the “infectious” alphavirus particle is a replicon particle(as described below) that can introduce the genomic RNA (i.e., replicon)into a host cell, is “replication-competent” to replicate the genomicRNA, but is “propagation-defective” in that it is unable to produce newalphavirus particles in the absence of helper sequences or a packagingcell that complements the deletions or other mutations in the replicon(i.e., provide the structural proteins that are not provided by thereplicon).

As used herein, the term “polypeptide” encompasses both peptides andproteins.

As used herein, an “isolated” nucleic acid (e.g., an “isolated DNA” oran “isolated genomic RNA”) means a nucleic acid separated orsubstantially free from at least some of the other components of thenaturally occurring organism or virus, for example, the cell or viralstructural components or other polypeptides or nucleic acids commonlyfound associated with the nucleic acid.

Likewise, an “isolated” polypeptide means a polypeptide that isseparated or substantially free from at least some of the othercomponents of the naturally occurring organism or virus, for example,the cell or viral structural components or other polypeptides or nucleicacids commonly found associated with the polypeptide.

As used herein, the terms “deleted” or “deletion” mean either totaldeletion of the specified segment or the deletion of a sufficientportion of the specified segment to render the segment inoperative ornonfunctional (e.g., does not encode a function protein), in accordancewith standard usage; See, e.g., U.S. Pat. No. 4,650,764 to Temin etal.).

The phrases “attenuating mutation” and “attenuating amino acid” as usedherein mean a nucleotide mutation or an amino acid encoded in view ofsuch mutation which result in a decreased probability of causing diseasein its host (i.e., a loss of virulence), in accordance with standardterminology in the art (See, e.g., B. Davis et al., Microbiology, 132(3d ed. 1980), whether the mutation be a substitution mutation or anin-frame deletion or insertion mutation. Attenuating mutations may be inthe coding or non-coding regions of the alphavirus genome. As known bythose skilled in the art, the phrase “attenuating mutation” excludesmutations or combinations of mutations which would be lethal to thevirus. Those skilled in the art will appreciate that some attenuatingmutations may be lethal in the absence of a second-site suppressormutation(s).

II. Alphavirus Vectors.

The present invention is practiced using alphavirus vectors, preferablya propagation-incompetent alphavirus vector, more preferably analphavirus replicon vector (as described below). Alphavirus and repliconvectors are described in U.S. Pat. No. 5,505,947 to Johnston et al.;U.S. Pat. No. 5,792,462 to Johnston et al., U.S. Pat. No. 5,814,482 toDubensky et al., U.S. Pat. No. 5,843,723 to Dubensky et al., U.S. Pat.No. 5,789,245 to Dubensky et al., U.S. Pat. No. 5,739,026 to Garoff etal., the disclosures of which are incorporated herein by reference intheir entireties. Typically, the alphavirus vector comprises one or moreheterologous nucleic acids. In embodiments of the invention at least oneof the heterologous nucleic acids encodes an antigen.

Alphavirus vectors can be transcribed in vitro from cDNA molecules, forexample, from a bacterial or viral promoter. Alternatively, they can beproduced in vivo from DNA, for example, from a viral or eukaryoticpromoter (see, e.g., U.S. Pat. Nos. 5,814,482 and 6,015,686;incorporated in their entireties herein by reference).

In particular embodiments of the invention, the alphavirus vector has aVEE virion shell. According to this embodiment, the alphavirus may be achimeric alphavirus and have a genomic RNA from another alphavirus.Alternatively, the alphavirus virion comprises a VEE E1 glycoprotein andmay comprise structural proteins (e.g., capsid and/or E2 glycoprotein)from other alphaviruses. In other embodiments, the alphavirus is a VEEvirus with both a VEE genomic RNA and virion coat.

Alphavirus vectors elicit a strong host response to the antigen(s)encoded by the heterologous sequence(s) in the vector. While not wishingto be held to any particular theory of the invention, it appears thatalphavirus vectors induce a more balanced and comprehensive immuneresponse (i.e., cellular and humoral immunity) than do conventionalvaccination methods. Moreover, it appears that alphavirus vectors inducea strong immune response, in part, because they directly infect andreplicate within dendritic cells. The resulting presentation of antigento the immune system induces a strong immune response. The alphavirus26S subgenomic promoter also appears to give high level of expression ofa heterologous nucleic acid encoding an immunogen.

The alphavirus vector preparation may be partially or highly purified,or may be a relatively crude cell lysate or supernate from a cellculture, as known in the art.

A. Double Promoter Vectors.

In one embodiment of the invention, the alphavirus genomic RNA is adouble promoter vector that is both replication and propagationcompetent. Double promoter vectors are described in U.S. Pat. Nos.5,185,440, 5,505,947 and 5,639,650, the disclosures of which areincorporated in their entireties by reference. In embodiments of theinvention, the alphavirus genomic RNA used to construct the doublepromoter vector is a VEE, Semliki Forest Virus, S.A.AR86, Girdwood S.A.,TR339, Sindbis or Ockelbo genomic RNA. In embodiments of the invention,the double promoter vector contains one or more attenuating mutations inthe genomic RNA. Attenuating mutations are described in more detailhereinbelow.

In particular embodiments, the double promoter vector is constructed soas to contain a second subgenomic promoter (i.e., 26S promoter) inserted3′ to the virus RNA encoding the structural proteins. The heterologousRNA is inserted between the second subgenomic promoter, so as to beoperatively associated therewith, and the 3′ UTR of the virus genome.Heterologous RNA sequences of less than about 3 kilobases, preferablythose less than about 2 kilobases, and more preferably those less thanabout 1 kilobase, can be inserted into the double promoter vector. Inone embodiment of the invention, the double promoter vector is derivedfrom a VEE genomic RNA, and the second subgenomic promoter is a VEEsubgenomic promoter. In an alternate embodiment, the double promotervector is derived from a Sindbis (e.g., TR339) genomic RNA, and thesecond subgenomic promoter is a Sindbis (e.g., TR339) subgenomicpromoter.

B. Replicon Vectors.

Replicon vectors, which are infectious, propagation-defective, virusvectors can also be used to carry out the present invention. Repliconvectors are described in more detail in WO 96/37616 to Johnston et al.,U.S. Pat. No. 5,505,947 to Johnston et al., and U.S. Pat. No. 5,792,462to Johnston et al; the disclosures of which are incorporated byreference herein in their entireties. Alphaviruses for constructing thereplicon vectors according to the present invention include, but are notlimited to, VEE, Semliki Forest Virus, S.A.AR86, Girdwood S.A., Sindbis(e.g., TR339), and Ockelbo.

In general, in the replicon system, one or more foreign gene(s) to beexpressed is/are inserted in place of at least a portion of one or moreof the viral structural protein genes in a transcription vectorcontaining the viral sequences necessary for viral replication (e.g.,the nsp1-4 genes). RNA transcribed from this vector contains sufficientviral sequences (e.g., the viral nonstructural genes) to be competentfor RNA replication and transcription. This RNA can be transcribed invitro or in vivo. In the case of in vitro transcribed RNA, the RNA isfirst transfected into susceptible cells by any method known in the art,wherein it is replicated and translated to give the replicationproteins. These proteins will transcribe the transfected RNA, includingthe transgene(s), which will, optionally, be translated. In certainembodiments, the transgene(s) is/are operatively associated with thealphavirus 26S subgenomic promoter, which will produce high level of thetranscript and, in the case of a translated RNA, the protein ofinterest. The autonomously replicating RNA (i.e., replicon) can only bepackaged into virus particles if the deleted alphavirus structuralprotein genes are provided. The deleted alphavirus structural proteingenes may be provided by any suitable means, e.g., by a stablytransformed packaging cell line (see, e.g., U.S. Pat. No. 5,789,245), orby one or more helper nucleic acid molecules (RNA or DNA), which areprovided to the cell along with the replicon vector, and are thenexpressed in the cell so that new replicon particles are produced in thecell.

In representative embodiments, the helper nucleic acids do not containthe viral nonstructural genes for replication, but these functions areprovided in trans by the replicon molecule. In one embodiment, thenon-structural proteins translated from the replicon molecule transcribethe structural protein genes on the helper nucleic acid molecule,resulting in the synthesis of viral structural proteins and packaging ofthe replicon into virus-like particles. As at least some of thealphavirus packaging or encapsidation signals are located within thenonstructural genes, the absence of these sequences in the helpernucleic acids precludes their incorporation into virus particles.

The replicon molecule is “propagation defective,” as describedhereinabove inasmuch as the replicon RNA in these particles does notinclude all of the alphavirus structural proteins required forencapsidation, at least a portion of at least one of the requiredstructural proteins being deleted therefrom. The replicon RNA thereforeonly initiates an abortive infection; no new viral particles areproduced, and there is no spread of the infection to other cells.

Typically, the replicon molecule comprises an alphavirus packagingsignal.

The replicon molecule is self-replicating. Accordingly, the repliconmolecule comprises sufficient coding sequences for the alphavirusnonstructural polyprotein so as to support self-replication. Inembodiments of the invention, the replicon encodes the alphavirus nsP1,nsP2, nsP3 and nsP4 proteins.

The replicon molecules of the invention “do not encode” one or more ofthe alphavirus structural proteins. By “do(es) not encode” one or morestructural proteins, it is intended that the replicon molecule does notencode a functional form of one or more structural proteins and, thus, acomplementing sequence is provided by a helper or packaging cell toproduce new virus particles. In embodiments of the invention, thereplicon molecule does not encode any of the alphavirus structuralproteins.

The replicon may not encode the structural protein(s) because the codingsequence is partially or entirely deleted from the replicon molecule.Alternatively, the coding sequence is otherwise mutated so that thereplicon does not express the functional protein. In embodiments of theinvention, the replicon lacks all or substantially all of the codingsequence of the structural protein(s) that is not encoded by thereplicon, e.g., so as to minimize recombination events with the helpersequences.

In particular embodiments, the replicon molecule may encode at leastone, but not all, of the alphavirus structural proteins. For example,the alphavirus capsid protein may be encoded by the replicon molecule.Alternatively, one or both of the alphavirus glycoproteins may beencoded by the replicon molecule. As a further alternative, the repliconmay encode the capsid protein and either the E1 or E2 glycoprotein.

In other particular embodiments, none of the alphavirus structuralproteins are encoded by the replicon molecule. For example, all oressentially all of the sequences encoding the alphavirus capsid proteinand glycoproteins may be deleted from the replicon molecule.

As yet another aspect, the invention provides a composition comprising apopulation of replicon particles containing no detectablereplication-competent alphavirus particles. Replication-competent virusmay be detected by any method known in the art, e.g., by neurovirulencefollowing intracerebral injection into suckling mice, or by passagetwice on alphavirus-permissive cells (e.g., BHK cells) and evaluationfor virus induced cytopathic effects.

III. Attenuating Mutations.

The present invention also provides alphavirus virion coats (e.g., VEEvirion coats) including attenuating mutations (as defined above) andgenomic RNA and DNA constructs encoding the same. Those skilled in theart will appreciate that the alphaviruses of the invention may furthercomprise attenuating mutations in the nonstructural protein codingregion or other regions of the alphavirus genome.

In particular embodiments, the attenuating mutation(s) reduces (e.g., byat least 25%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) theneurovirulence of the alphavirus vector (e.g., as determined byintracerebral injection in weanling or adult mice). It is not necessarythat the attenuating mutations of the invention eliminate all pathologyor adverse effects associated with virus administration, as long asthere is some improvement or benefit (e.g., increased safety and/orreduced morbidity and/or reduced mortality) as a result of theattenuating mutation.

Appropriate attenuating mutations will be dependent upon the alphavirusused. Exemplary attenuating mutations include, but are not limited to,those described in U.S. Pat. No. 5,505,947 to Johnston et al., U.S. Pat.No. 5,185,440 to Johnston et al., U.S. Pat. No. 5,643,576 to Davis etal., U.S. Pat. No. 5,792,462 to Johnston et al., and U.S. Pat. No.5,639,650 to Johnston et al., the disclosures of which are incorporatedherein in their entirety by reference.

Other attenuating mutations of particular interest include attenuatingmutations in the E1 glycoprotein of the alphavirus virion shell (e.g.,VEE virion shell). While not wishing to be bound by any theory of theinvention, the E2 glycoprotein is believed to bind to cellular virusreceptors and, thus, E1 mutants may advantageously achieve attenuationwithout disrupting cellular targeting. Accordingly, in embodiments ofthe invention, the attenuating mutation is a mutation in the E1glycoprotein (e.g., the VEE E1 glycoprotein) that does not undulyinterfere (e.g., reduce by more than 25%, 35% or 50%) with cellulartargeting, receptor binding and/or infectivity, for example, to or indendritic cells.

In other particular embodiments of the invention, the attenuatingmutation is in the putative fusogenic peptide region in the alphavirusE1 glycoprotein (e.g., the fusogenic peptide region of the VEE E1glycoprotein). This region is from about amino acid 80 to about aminoacid 93 of the E1 glycoprotein and contains a stretch of uncharged andhydrophobic amino acids (see, e.g., Davis et al., (1994) Arch Virol[Suppl.] 9:99). Following virus binding via the E2 glycoprotein to thecell surface receptor, the glycoproteins rearrange and this hydrophobicdomain is exposed and is believed to facilitate entry of the virusacross the cellular membrane.

In particular embodiments, the alphavirus virion shell has anattenuating mutation at E1 glycoprotein amino acid position 81. Forexample, the attenuating mutation may be a phenylalanine to leucine orisoleucine mutation in Sindbis virus (e.g., strain TR339) or a mutationfrom tyrosine to leucine or isoleucine in Semliki Forest Virus or RossRiver Virus. Similar mutations in the E1 fusogenic region may be made inany alphavirus (as defined above).

In other embodiments, the alphavirus comprises a VEE virion shellcomprising an attenuating mutation at E1 glycoprotein amino acidposition 81 and/or 253. The VEE virion shell may additionally containother attenuating mutations. Attenuating mutations may be selected asdescribed below. In particular embodiments, the attenuating mutation atamino acid position 81 is a mutation from phenylalanine to leucine orisoleucine. In other particular embodiments, the attenuating mutation atamino acid position 253 is a mutation from phenylalanine to serine orthreonine.

Another particular attenuating mutation is an attenuating mutation inthe VEE virion shell at E1 amino acid position 83. Typically, thisattenuating mutation is used together with a second site suppressormutation to avoid lethality.

One barrier encountered with many attenuating mutations is that theattenuated virus frequently has decreased immunogenicity, i.e., thevirus is safer, but is less efficacious in eliciting the desired immuneresponse. The present invention advantageously provides immunogeniccompositions comprising attenuated alphavirus particles with improvedefficacy (e.g., provides protection at a lower dosage) as compared withother attenuated alphaviruses. Methods of assessing the effectiveness ofimmunogenic compositions are well known in the art and include but arenot limited to methods of evaluating protection against a challengepathogen and indirect methods such as determination of antibody titers.

Thus, in particular embodiments, the present invention providesalphaviruses having attenuating mutations that achieve attenuationwithout significantly reducing (e.g., by more than 25%, 35% or 50%)immunogenicity, thereby resulting in a need for a corresponding increasein dosage. In particular embodiments, the present invention provides anattenuated alphavirus having a VEE shell, where the alphavirus issubstantially as immunogenic as, or is even substantially moreimmunogenic than, a comparable alphavirus having a wild-type VEE virionshell (for example, the VEE 3000 described herein), i.e., asubstantially similar number of infectious virus particles or evensubstantially less virus is required to provide an immunogenicallyeffective dosage. By “substantially as immunogenic” it is intended thatthe attenuated alphavirus is as immunogenic as an alphavirus having awild-type VEE coat (e.g., VEE 3000) at a dosage that is about 50% to200% of the dosage of the virus having the wild-type VEE coat, i.e.,one-half to two times as much attenuated virus is needed to elicit thesame immune response as an alphavirus having a wild-type coat. In otherembodiments, the alphavirus is “substantially more immunogenic” than acomparable alphavirus comprising a wild-type VEE coat, i.e., asubstantially lower dosage (e.g., less than about 50%) of the attenuatedvirus provides the same immune response as the alphavirus comprising thewild-type VEE coat.

By “substantially less immunogenic” it is intended that the attenuatedalphavirus is as immunogenic as an alphavirus having a wild-type VEEcoat (e.g., VEE 3000) at a dosage that is about 250% or more of thedosage of a comparable alphavirus having a wild-type VEE coat, i.e.,2.5-times or more attenuated virus is needed to elicit the same immuneresponse as an alphavirus having a wild-type coat. Because of the safetybenefits of an attenuated virus, the concerns relating to administeringhigh virus dosages to subjects, and the costs of virus production,alphaviruses having attenuated VEE coats that are less immunogenicallyeffective than a comparable alphavirus having a wild-type VEE virionshell can nonetheless be advantageous and are encompassed by the presentinvention, e.g., attenuated viruses that require a dosage that is lessthan about 5ive-fold, less than about 7.5-fold, less than about 10-fold,less than about 15-fold, less than about 25-fold, less than about50-fold higher, or even less than about 100-fold higher than the dosageof a comparable virus having a wild-type VEE virion shell to elicit asimilar immune response.

Alternatively stated, in other embodiments of the invention, theattenuated virus is more immunogenic than a comparable attenuated viruscomprising the 3014 VEE coat described below, i.e., a lower dosage ofthe attenuated virus of the invention produces an immunogenicallyeffective response as compared with the dosage of an alphaviruscomprising the 3014 coat. In particular embodiments, the immunogenicallyeffective dosage of the attenuated virus of the invention is less thanabout 25%, about 50%, or about 75% of the dosage of a comparable virushaving a 3014 VEE virion shell. In other embodiments, theimmunogenically effective dosage of the attenuated virus is reduced byabout one order of magnitude, two orders of magnitude, or even threeorders of magnitude or more as compared with the dosage of a comparablevirus having a 3014 VEE coat.

Those skilled in the art will appreciate that the relativeimmunogenicity of the attenuated alphavirus as compared with a suitablenon-attenuated control virus (e.g., having a VEE 3000 coat) may varydepending upon the particular dosage, route of administration, speciesand age of the subject, and the like.

As described in Bernard et al., (2000) Virology 276:93, the wild typeVEE virion shell only interacts poorly with heparin, whereas someattenuated VEE mutants (e.g., the 3014 mutant having an Ala→Thr mutationat E1 position 272, a Glu→Lys mutation at E2 position 209, and a Ile→Asnmutation at E2 position 239) bind relatively strongly to heparin.Methods of detecting viral interaction with heparin are known to thoseskilled in the art, for example, binding to immobilized heparin (e.g., aheparin column or beads) or inhibition of cell infectivity or binding byheparin (e.g., to BHK cells or dendritic cells), which are described inBernard et al., (2000) Virology 276:93).

In some embodiments, the attenuated viruses of the invention do notexhibit detectable binding to, or only weakly bind to, heparin orheparan sulfate. According to these embodiments, the attenuated virusesof the invention are more similar to the wild-type virus than the 3014mutant described above with respect to heparin binding. While notwishing to be bound by any particular theory, it appears that binding toheparin and/or heparan sulfate may increase viral clearance rates andreduce infectivity, with a resulting loss of immunogenicity. In otherparticular embodiments of the invention, the attenuated virus (e.g., anattenuated alphavirus with a VEE virion shell) does not exhibitdetectable binding to glycosaminoglycans (e.g., heparin, heparansulfate, chondroitin, chondroitin sulfate and/or dextran sulfate) oronly exhibits weak binding thereto. Particular alphaviruses withattenuating mutations in the E2 glycoprotein and having only weakbinding to heparin have been described by Bernard et al., (2000)Virology 276:93, the disclosures of which are incorporated by referenceherein in its entirety.

In particular embodiments, the alphavirus comprises a VEE virion shellcomprising an attenuating mutation in the E1 glycoprotein, where thealphavirus exhibits no detectable binding or only weak binding toheparin. In other embodiments, the alphavirus comprises a VEE virionshell comprising an attenuating mutation in the fusogenic peptide regionof the E1 glycoprotein (as described above), wherein the alphavirusexhibits no detectable binding or only weak binding to heparin. Thevirion shell can further comprise additional attenuating mutations inthe E2 and/or E3 glycoproteins (exemplary mutations in the E2 and E3glycoproteins are discussed below).

In representative embodiments of the invention, the alphavirus comprisesa VEE virion shell comprising an attenuating mutation at E1 amino acidposition 81 and/or E1 253 (each as described above), and exhibits nodetectable binding or only weak binding to heparin. For example, the3042 mutation has a Phe→lie mutation at E1 position 81. Alternatively oradditionally, the alphavirus comprises a VEE coat comprising anattenuating mutation that results in the deletion of the furin cleavagesite in the E3 glycoprotein (e.g., deletion of E3 amino acids 56-59),and exhibits no detectable binding or only weak binding to heparin. Thistype of attenuating mutation may be present in conjunction with a secondsite mutation to maintain viability (e.g., a second site mutation at E1amino acid position 253). Thus, in one particular embodiment, theattenuated mutant comprises a mutation (e.g., Phe→Ser) at E1 position253 and a deletion of the furin cleavage site (e.g., deletion of E3amino acids 56-59), and exhibits no detectable binding or only weakbinding to heparin.

In still other embodiments, the attenuated alphavirus comprises a VEEvirion shell comprising an attenuating mutation at E1 amino acid 272(e.g., an Ala→Thr mutation). In further embodiments, the attenuatedalphavirus comprises a VEE virion shell comprising attenuating mutationsat E2 amino acids 76 and 166 (e.g., Glu→Lys mutation at E2 position 76and a Lys→Glu mutation at E2 position 116).

As noted above, virus interaction with heparin may be assessed byinhibition of virus infectivity. In particular embodiments, a virus that“exhibits (only) weak binding” to heparin does not demonstrate asubstantial reduction (i.e., more than about 50%) in infectivity (e.g.,in BHK cells or dendritic cells) in the presence of relatively lowconcentrations of heparin (e.g., concentrations of about 50, 100, 150 or200 μg/ml or less). In particular embodiments, heparin binds to theattenuated virus comprising the VEE virion shell (e.g., interfering withinfectivity of the virus) with an affinity that is similar to or lessthan the affinity of heparin for the wild-type virus or, alternatively,is less than about two-fold, three-fold, four-fold, or five-fold greaterthan the affinity of the wild-type virion shell for heparin.Alternatively stated, in other embodiments, by “exhibits (only) weakbinding” to heparin, it is meant that the affinity of heparin binding tothe alphavirus comprising the attenuated VEE virion shell is less thanabout 25%, 20%, 15%, 10%, 5% or less than the affinity of the 3014 coatfor heparin, e.g., interference of virus infectivity by heparin is lessthan about 25%, 20%, 15%, 10%, 5% or less than the interference ofinfectivity by a virus comprising the 3014 coat.

One of ordinary skill in the art may routinely identify attenuatingmutations other than those specifically disclosed herein using methodsknown to those skilled in the art (see, e.g., Olmsted et al., (1984)Science 225:424 and Johnston and Smith (1988) Virology 162:437). Olmstedet al. describes a method of identifying attenuating mutations inSindbis virus by selecting for rapid growth in cell culture. TheJohnston and Smith publication describes the identification ofattenuating mutations in VEE by applying direct selective pressure foraccelerated penetration of BHK cells.

Likewise, one of ordinary skill in the art may routinely identifyattenuating mutations having the desired characteristics (for example,improved immunogenicity as compared with known attenuating alphaviruses)using techniques for assessing immunogenicity known in the art (e.g.,antibody titers may be measured by ELISA assay, hemagglutinininhibition, virus neutralization and plaque reduction neutralizationassays) and as described in the working examples herein.

The present invention also includes methods for identification ofattenuating mutations that lack the ability to bind heparin and haveincreased immunogenicity. One such method involves the selection ofvirus particles with the ability to infect cell monolayers in vitro inthe presence of heparin or heparan sulfate. In other embodiments of thismethod, other glycosaminoglycans can be used for this selection,including, but not limited to dextran sulfate, chondroitin sulfate A,chondroitin sulfate B as described in Klimstra et al. (1998) J. Virol.72:7357-7366.

A spectrum of mutations are first engineered into the E1 and/or E2glycoproteins of the alphavirus by methods well known in the art, suchas random, site-directed or saturation mutagenesis. This heterogeneouspopulation of mutated viral particles is then incubated with apermissive (i.e. a cell line that can be infected by the alphavirus)cell line in vitro in the presence of glycosaminoglycans at a sufficientconcentration as to be inhibitory to the infection of the cell line byviral particles known to bind heparin, e.g., between 20 and 300microgram/per ml. Alternatively, the viral population can be incubatedwith the glycosaminoglycan prior to exposure of the cell line to themutant particles. This screening method selectively prohibits the entryof viral particles with significant affinity for the particularglycosaminoglycan and imposes selective pressure, allowingidentification of low or non-binding glycosaminoglycan mutants that areable to enter the cell and establish a productive infection. Thesemutants are then passed for multiple passages through the cell line,under the same or increased stringencies of selection fornon-glycosaminoglycan binding alphaviral shells. The selected mutantpopulations are isolated by plaque assay, plaque purified by methodsknown in the art to produce clonal populations of viral particles thatare sequenced to identify individual and/or combinations ofnon-glycosaminoglycan binding mutations. These mutations, eitherseparately or in combination, are introduced into the wild-type virusand further selected for their attenuation and potential increasedimmunogenicity by methods known in the art, e.g. Davis et al. 1991; U.S.Pat. No. 5,185,440; U.S. Pat. No. 5,505,947.

Another method for selecting attenuating mutations encompassed by thisinvention is to take the mutagenized viral population described above,which consists of a mixed population of alphaviral shell-mutatedviruses, and select within this population using affinity-basedchromatographic techniques, for example glycosaminoglycan matrixchromatographic columns (specifically heparin or any otherglycosaminoglycan as described above). Low ornon-glycosaminoglycan-binding mutant virus particles will pass throughor elute from the column in the early fractions. Individual clonal viralpopulations are then isolated from these fractions by plaquepurification. Purified viral clones are sequenced by standard methods toidentify the specific mutations that can be introduced into thewild-type virus shell, and virus or replicon particles made with suchmutated shells are assayed for both attenuation and immunogenicity. Theoverall stringency of this column selection method can be increased ordecreased by methods known in the art such as altering columnconditions, e.g. buffer pH, salt concentration, column length, andchromatographic matrix choice, to optimize the retention ofglycosaminoglycan binding mutants and to expand the range of mutationsthat might be usefully employed in this invention.

Accordingly, the present invention encompasses other attenuatingmutations that do not substantially reduce immunogenicity (i.e., theattenuated virus is essentially as immunogenic as, or more immunogenicthan, a comparable alphavirus having a wild-type coat).

When the alphavirus structural proteins are from VEE, other suitableattenuating mutations may be selected from the group consisting ofcodons at E2 amino acid position 76 which specify an attenuating aminoacid, preferably lysine, arginine, or histidine as E2 amino acid 76;codons at E2 amino acid position 120 which specify an attenuating aminoacid, preferably lysine as E2 amino acid 120; codons at E2 amino acidposition 209 which specify an attenuating amino acid, preferably lysine,arginine or histidine as E2 amino acid 209; codons at E1 amino acid 272which specify an attenuating amino acid, preferably threonine or serineas E1 amino acid 272, as provided above.

Other suitable attenuated alphavirus vectors comprise an attenuatingmutation in the capsid protease that reduces, preferably ablates, theautoprotease activity of the capsid and results, therefore, innon-viable virus. Capsid mutations that reduce or ablate theautoprotease activity of the alphavirus capsid are known in the art, seee.g., WO 96/37616 to Johnston et al., the disclosure of which isincorporated herein in its entirety. In particular embodiments, thealphavirus vector comprises a VEE capsid protein in which the capsidprotease is ablated, e.g., by introducing an amino acid substitution atVEE capsid position 152, 174, or 226. Alternatively, one or more of thehomologous positions in other alphaviruses may be altered to reducecapsid protease activity.

If the alphavirus vector comprises a Sindbis-group virus (e.g., Sindbis,S.A.AR86, GirdwoodSA, Ockelbo) capsid protein, the attenuating mutationmay be a mutation at capsid amino acid position 215 (e.g., a Ser→Ala)that reduces capsid autoprotease activity (see, Hahn et al., (1990) J.Virology 64:3069).

In some embodiments, the alphavirus structural proteins are fromS.A.AR86. Exemplary attenuating mutations in the S.A.AR86 structuralproteins are known in the art (see, e.g., International Application No.PCT/US03/09121; incorporated by reference herein in its entirety).

To identify attenuating mutations other than those specificallydisclosed herein, amino acid substitutions may be based on anycharacteristic known in the art, including the relative similarity ordifferences of the amino acid side-chain substituents, for example,their hydrophobicity, hydrophilicity, charge, size, and the like.

Amino acid substitutions other than those disclosed herein may beachieved by changing the codons of the genomic RNA sequence (or a DNAsequence), according to the following codon table: TABLE 1 Amino AcidsCodons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Asparticacid Asp D GAC GAU Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUCUUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine IleI AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUUMethionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCGCCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGUSerine Ser S AGC ACU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACUValine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

In identifying other attenuating mutations, the hydropathic index of theamino acids may be considered. The importance of the hydropathic aminoacid index in conferring interactive biologic function on a protein isgenerally understood in the art (see, Kyte and Doolittle, (1982) J. Mol.Biol. 157:105; incorporated herein by reference in its entirety). It isaccepted that the relative hydropathic character of the amino acidcontributes to the secondary structure of the resultant protein, whichin turn defines the interaction of the protein with other molecules, forexample, enzymes, substrates, receptors, DNA, antibodies, antigens, andthe like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,Id.), these are:

isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

Accordingly, the hydropathic index of the amino acid (or amino acidsequence) may be considered when identifying additional attenuatingmutations according to the present invention.

It is also understood in the art that the substitution of amino acidscan be made on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(incorporated herein by reference in its entirety) states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (±3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±l); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Thus, the hydrophilicity of the amino acid (or amino acid sequence) maybe considered when identifying additional attenuating mutationsaccording to the present invention.

The attenuating mutations may be located in any of the structuralproteins. The alphavirus vectors may contain two or more attenuatingmutations within one structural protein or may contain two or moreattenuating mutations distributed among the structural proteins.Further, additional attenuating mutations may be located on the repliconRNA in either the non-structural or structural coding regions as well asin non-coding regions.

Mutations may be introduced into the alphavirus vector by any methodknown in the art. For example, mutations may be introduced into thealphavirus RNA by performing site-directed mutagenesis on the cDNA whichencodes the RNA, in accordance with known procedures (see, Kunkel, Proc.Natl. Acad. Sci. USA 82,488 (1985), the disclosure of which isincorporated herein by reference in its entirety). Alternatively,mutations may be introduced into the RNA by replacement of homologousrestriction fragments in the cDNA which encodes for the RNA, inaccordance with known procedures.

IV. Helper Cells, Helper Constructs and Methods of Producing ViralParticles.

Other aspects of the present invention are methods and helper cells forproducing alphavirus particles in vitro. Methods and helper cells forproducing alphavirus stocks, including double-promoter alphaviruses andalphavirus replicon particles are known in the art. See, e.g., U.S. Pat.No. 5,185,440 to Davis et al., U.S. Pat. No. 5,505,947 to Johnston etal.; U.S. Pat. No. 5,792,462 to Johnston et al., and Pushko et al.(1997) Virol. 239:389-401; the disclosures of which are incorporatedherein by reference in their entireties. Methods for producingalphavirus particles using stably transformed packaging cell linesand/or DNA-based vector launches, such as the “ELVIS” system are alsoknown in the art (see, e.g., U.S. Pat. No. 5,814,482 to Dubensky et al.,U.S. Pat. No. 5,843,723 to Dubensky et al., U.S. Pat. No. 5,789,245 toDubensky et al.; incorporated herein by reference in their entireties).

In representative embodiments, the methods and helper cells are used toproduce propagation-incompetent alphavirus particles, for example,propagation-incompetent alphavirus replicon particles. According to thisembodiment, the helper cells of the invention contain one or more helpernucleic acid sequences (e.g., as DNA and/or RNA molecules) encoding thealphavirus structural proteins (e.g., VEE structural proteins). Thecombined expression of the replicon molecule and the one or more helpermolecules in the helper cell results in the production of an assembledalphavirus particle comprising a replicon RNA packaged within a virioncomprising alphavirus structural proteins, which is able to infect acell, but is unable to produce a productive infection (i.e., produce newvirus particles).

In embodiments of the invention, the population of alphavirus particlesproduced according to the invention contains no detectablepropagation-competent alphavirus particles. Propagation-competent virusmay be detected by any method known in the art, e.g., by neurovirulencefollowing intracerebral injection into suckling mice, or-by passagetwice on alphavirus-permissive cells (e.g., BHK cells) and evaluationfor virus induced cytopathic effects.

The helper cells are typically alphavirus-permissive cells.Alphavirus-permissive cells employed in the methods of the presentinvention are cells that, upon transfection with the viral RNAtranscript, are capable of producing viral particles. Alphaviruses havea broad host range. Examples of suitable host cells include, but are notlimited to fibroblasts, Vero cells, baby hamster kidney (BHK) cells, 293cells, 293T cells, and chicken embryo fibroblast cells (e.g., DF-1cells).

In particular embodiments, the helper cells of the invention maycomprise sequences encoding the alphavirus structural proteinssufficient to produce an alphavirus particle, as described herein.Alternatively, or additionally, the helper cell may comprise a repliconRNA comprising one or more heterologous sequences, also as describedherein.

As described hereinabove, in the production of a replicon particle,sequences encoding the alphavirus structural proteins are distributedamong one or more helper molecules (preferably, two or three helper RNAsor DNAs). In addition, one or more structural proteins may be encoded bythe replicon RNA, provided that the replicon RNA does not encode atleast one structural protein such that the resulting alphavirus particleis propagation-incompetent in the absence of the helper sequence(s).

According to the present invention, at least one of the alphavirusstructural and/or non-structural proteins encoded by the replicon andhelper molecules contain one or more attenuating mutations, as describedherein.

In one particular embodiment, the replicon molecule encodes at leastone, but not all, of the alphavirus structural proteins (e.g., the E1and/or E2 glycoproteins and/or the capsid protein). In one particularembodiment, the replicon encodes the capsid protein, and the E1 and E2glycoproteins are encoded by one or more separate helper molecules. Itmay be advantageous to provide the glycoproteins by two separate helpermolecules, so as to minimize the possibility of recombination to producereplication-competent virus.

In another embodiment, the replicon does not encode any of the E1glycoprotein, the E2 glycoprotein, or the capsid protein. According tothis embodiment, the capsid protein and alphavirus glycoproteins areencoded by one or more helper molecules, preferably two or more helpermolecules. By distributing the coding sequences for the structuralproteins among two, three or even more helper molecules, the likelihoodthat recombination will result in replication-competent virus isreduced.

In a further embodiment, the replicon does not encode any of thealphavirus structural proteins, and may lack the sequences encoding thealphavirus structural proteins.

As described above, the replicon may not encode the structuralprotein(s) because of a partial or complete deletion of the codingsequence(s) or otherwise contains a mutation that prevents theexpression of a functional protein(s). In embodiments of the invention,all or substantially all of the coding sequences for the structuralprotein(s) that is not encoded by the replicon are deleted from thereplicon molecule.

In one embodiment, the E1 and E2 glycoproteins are encoded by one helpermolecule, and the capsid protein is encoded by another helper molecule.In another preferred embodiment, the E1 glycoprotein, E2 glycoprotein,and capsid protein are each encoded by separate helper molecules. Inother embodiments, the capsid protein and one of the glycoproteins areencoded by one helper molecule, and the other glycoprotein is encoded bya second helper molecule.

In other particular embodiments, the helper and replicon sequences areRNA molecules that are introduced into the cell, e.g., by lipofection orelectroporation. Uptake of helper RNA and replicon RNA molecules intopackaging cells in vitro can be carried out according to any suitablemeans known to those skilled in the art. Uptake of RNA into the cellscan be achieved, for example, by treating the cells with DEAE-dextran,treating the RNA with LIPOFECTIN™ before addition to the cells, or byelectroporation, with electroporation being the currently preferredmeans. These techniques are well known in the art. See e.g., U.S. Pat.No. 5,185,440 to Davis et al., and PCT Publication No. WO 96/37616 toJohnston et al., the disclosures of which are incorporated herein byreference in their entirety.

Alternatively, one or all of the helper and/or replicon molecules areDNA molecules, which are either stably integrated into the genome of thehelper cell or expressed from an episome (e.g., an EBV derived episome).The DNA molecule may be any vector known in the art, including but notlimited to a non-integrating DNA vector, such as a plasmid, or a viralvector.

V. Recombinant Alphavirus Vectors.

According to embodiments of the invention, it is desirable to employ analphavirus vector that encodes one or more (e.g., two, three, four,five, etc.) heterologous nucleic acid sequences, preferably eachencoding an antigen according to the present invention. In particularembodiments, wherein there are two or more heterologous nucleotidesequences, each heterologous nucleic acid sequence will typically beoperably associated with a promoter.

Alternatively, an internal ribosome entry site (IRES) sequence(s) can beplaced downstream of the first heterologous nucleic acid sequence andupstream of a second or additional heterologous nucleic acidsequence(s). In any of these embodiments, the heterologous nucleic acidsequence(s) can be associated with a constitutive or inducible promoter.An exemplary promoter is an alphavirus 26S subgenomic promoter (e.g.,VEE 26S subgenomic promoter). In general, the S.A.AR86 26S subgenomicpromoter can be used with S.A.AR86 replication proteins, and the VEE 26Ssubgenomic promoter can be used with VEE replication proteins, and thelike.

Heterologous nucleic acids of interest include nucleic acids encodingpeptides and proteins, including immunogenic (e.g., for an immunogeniccomposition or a vaccine) or therapeutic (e.g., for medical orveterinary uses) polypeptides.

An “immunogenic” polypeptide, or “immunogen” as used herein is anypolypeptide that elicits an immune response in a subject, and inparticular embodiments, the immunogenic polypeptide is suitable forproviding some degree of protection to a subject against a disease. Thepresent invention may be employed to express an immunogenic polypeptidein a subject (e.g., for vaccination) or for immunotherapy (e.g., totreat a subject with cancer or tumors).

An immunogenic polypeptide, or immunogen, may be any polypeptidesuitable for protecting the subject against a disease, including but notlimited to microbial, bacterial, protozoal, parasitic, and viraldiseases. For example, the immunogen may be an orthomyxovirus immunogen(e.g., an influenza virus immunogen, such as the influenza virushemagglutinin (HA) surface protein or the influenza virus nucleoproteingene, or an equine influenza virus immunogen), or a lentivirus immunogen(e.g., an equine infectious anemia virus immunogen, a SimianImmunodeficiency Virus (SIV) immunogen, or a Human ImmunodeficiencyVirus (HIV) immunogen, such as the HIV or SIV envelope GP160 protein,the HIV or SIV matrix/capsid proteins, and the HIV or SIV gag, pol, ref,tat, nef and env genes products). The immunogen may also be anarenavirus immunogen (e.g., Lassa fever virus immunogen, such as theLassa fever virus nucleocapsid protein gene and the Lassa fever envelopeglycoprotein gene), a Picornavirus immunogen (e.g., a Foot and MouthDisease virus immunogen), a poxvirus immunogen (e.g., vaccinia, such asthe vaccinia L1 or L8 genes), an Orbivirus immunogen (e.g., an Africanhorse sickness virus immunogen), a flavivirus immunogen (e.g., a yellowfever virus immunogen, a West Nile virus immunogen, or a Japaneseencephalitis virus immunogen), a filovirus immunogen (e.g., an Ebolavirus immunogen, or a Marburg virus immunogen, such as NP and GP genes),a bunyavirus immunogen (e.g., RVFV, CCHF, and SFS immunogens), or acoronavirus immunogen (e.g., an infectious human coronavirus immunogen,such as the human coronavirus envelope glycoprotein gene, or a porcinetransmissible gastroenteritis virus immunogen, or an avian infectiousbronchitis virus immunogen). The immunogen may further be a polioantigen, tuberculosis antigen, herpes antigen (e.g., CMV, EBV, HSVantigens) mumps antigen, measles antigen, rubella antigen, diptheriatoxin or other diptheria antigen, pertussis antigen, hepatitis (e.g.,hepatitis A or hepatitis B) antigen, or any other vaccine antigen knownin the art.

In embodiments of the invention, the antigen is Simian ImmunodeficiencyVirus (SIV) or Human Immunodeficiency Virus (HIV) antigen. For example,the antigen may be the product(s) of the SIV or HIV gag, env, ref, tat,nef or pol genes, or combinations thereof. In other particularembodiments, the antigen(s) is/are from a specific lade of the HIVvirus, e.g., Clade B, C or E or combinations thereof.

Accordingly, in particular embodiments, the subject is a human subjector a simian subject that is infected with, or is at risk of becominginfected with HIV or SIV, respectively. Likewise, in other embodiments,the subject is a human subject that has, or is at risk of developing,AIDs.

The present invention may also be advantageously employed to produce animmune response against chronic or latent infective agents, whichtypically persist because they fail to elicit a strong immune responsein the subject. Illustrative latent or chronic infective agents include,but are not limited to, hepatitis B, hepatitis C, Epstein-Barr Virus,herpes viruses, human immunodeficiency virus, and human papillomaviruses. Alphavirus vectors encoding antigens from these infectiousagents may be administered to a cell or a subject according to themethods described herein.

Alternatively, the immunogen may be any tumor or cancer antigen.Preferably, the tumor or cancer antigen is expressed on the surface ofthe cancer cell. Exemplary cancer antigens for specific breast cancersare the HER2 and BRCA1 antigens. Other illustrative cancer and tumorcell antigens are described in S. A. Rosenberg; (1999) Immunity 10:281)and include, but are not limited to: MART-1/MelanA, gp100, tyrosinase,TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4,β-catenin, MUM-1, Caspase-8, KIAA0205, HPVE&, SART-1, PRAME, p15, andp53 antigens, and epitopes or fragments thereof. Additional cancerimmunogens are the prostate-specific membrane antigen (PSMA), theprostate-specific antigen (PSA), CEA, or epitopes thereof.

The immunogen may also be a “universal” or “artificial” cancer or tumorantigen as described in international patent publication WO 99/51263,which is hereby incorporated by reference in its entirety.

The term “cancer” has its understood meaning in the art, for example, anuncontrolled growth of tissue that has the potential to spread todistant sites of the body (i.e., metastasize). Exemplary cancersinclude, but are not limited to, leukemias, lymphomas, colon cancer,renal cancer, liver cancer, breast cancer, lung cancer, prostate cancer,ovarian cancer, melanoma, and the like. Other illustrative cancersinclude cancers of the bone and bone marrow. Also encompassed aremethods of treating and preventing tumor-forming cancers. The term“tumor” is also understood in the art, for example, as an abnormal massof undifferentiated cells within a multicellular organism. Tumors can bemalignant or benign. Preferably, the methods disclosed herein are usedto prevent and treat malignant tumors.

Cancer and tumor antigens according to the present invention have beendescribed hereinabove. Alphaviruses encoding cancer or tumor antigensmay be administered in methods of treating cancer or tumors,respectively.

By the terms “treating cancer” or “treatment of cancer”, it is intendedthat the severity of the cancer is reduced or the cancer is at leastpartially eliminated. These terms may also indicate that metastasis ofthe cancer is reduced or at least partially eliminated. By the terms“prevention of cancer” or “preventing cancer” it is intended that themethods at least partially eliminate or reduce the incidence or onset ofcancer. Alternatively stated, the present methods slow, control,decrease the likelihood or probability, or delay the onset of cancer inthe subject.

Likewise, by the terms “treating tumors” or “treatment of tumors”, it isintended that the severity of the tumor is reduced or the tumor is atleast partially eliminated. These terms may also indicate thatmetastasis of the tumor is reduced or at least partially eliminated. Bythe terms “prevention of tumors” or “preventing tumors” it is intendedthat the inventive methods at least partially eliminate or reduce theincidence or onset of tumors. Alternatively stated, the present methodsslow, control, decrease the likelihood or probability, or delay theonset of tumors in the subject.

It is known in the art that immune responses may be enhanced byimmunomodulatory cytokines (e.g., a-interferon, β-interferon,γ-interferon, ω-interferon, τ-interferon, interleukin-1α,interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin5, interleukin-6, interleukin-7, interleukin-8, interleukin-9,interleukin-10, interleukin-11, interleukin 12, interleukin-13,interleukin-14, interleukin-18, B cell Growth factor, CD40 Ligand, tumornecrosis factor-α, tumor necrosis factor-β, monocyte chemoattractantprotein-1, granulocyte-macrophage colony stimulating factor, andlymphotoxin). Accordingly, in particular embodiments of the invention,immunomodulatory cytokines (e.g., CTL inductive cytokines) areadministered to a subject in conjunction with the methods describedherein for producing an immune response or providing immunotherapy.

Cytokines may be administered by any method known in the art. Exogenouscytokines may be administered to the subject, or alternatively, anucleotide sequence encoding a cytokine may be delivered to the subjectusing a suitable vector, and the cytokine produced in vivo. In preferredembodiments, an alphavirus vector encoding a cytokine is used to deliverthe cytokine to the subject.

The present invention further finds use in methods of producingantibodies in vivo for passive immunization techniques. According tothis embodiment, an alphavirus vector expressing an immunogen ofinterest is administered to a subject, as described herein by directadministration or ex vivo cell manipulation techniques. The antibody maythen be collected from the subject using routine methods known in theart. The invention further finds use in methods of producing antibodiesagainst an immunogen expressed from an alphavirus vector for any otherpurpose, e.g., for diagnostic purpose or for use in histologicaltechniques.

The heterologous nucleic acid may be operably associated with expressioncontrol elements, such as transcription/translation control signals,origins of replication, polyadenylation signals, and internal ribosomeentry sites (IRES), promoters, enhancers, and the like. Those skilled inthe art will appreciate that a variety of promoter/enhancer elements maybe used depending on the level and tissue-specific expression desired.The promoter/enhancer may be constitutive or inducible, depending on thepattern of expression desired. The promoter/enhancer may be native orforeign and can be a natural or a synthetic sequence.

Promoters/enhancers that are native to the subject to be treated aremost preferred. Also preferred are promoters/enhancers that are nativeto the heterologous nucleic acid sequence. The promoter/enhancer ischosen so that it will function in the target cell(s) of interest.Mammalian promoters/enhancers are also preferred.

Preferably, the heterologous nucleotide sequence is operably associatedwith a promoter that provides high level expression of the heterologousnucleotide sequence, e.g., an alphavirus subgenomic 26S promoter (inparticular, a VEE 26S subgenomic promoter).

In embodiments of the invention in which the heterologous nucleic acidsequence(s) will be transcribed and then translated in the target cells,specific initiation signals are generally required for efficienttranslation of inserted protein coding sequences. These exogenoustranslational control sequences, which may include the ATG initiationcodon and adjacent sequences, can be of a variety of origins, bothnatural and synthetic.

VI. DNA Sequences, Vectors and Transformed Cells.

As a further aspect, the present invention provides DNA sequences (e.g.,cDNA sequences) and vectors encoding infectious recombinant alphavirusgenomic RNA transcripts (e.g., VEE genomic RNA transcripts) according tothe present invention, comprising one or more heterologous nucleotidesequences. Also provided are alphavirus particles containing therecombinant alphavirus genomic RNA transcribed from the DNA molecules.

The present invention further provides vectors comprising a DNA sequenceencoding a recombinant alphavirus genomic RNA transcript operablyassociated with a promoter that drives transcription of the DNAsequence. Examples of promoters which are suitable for use with the DNAsequences of the present invention include, but are not limited to T3promoters, T7 promoters, cytomegalovirus (CMV) promoters, and SP6promoters.

The DNA sequence may be encoded by any suitable vector known in the art,including but not limited to, plasmids, naked DNA vectors, yeastartificial chromosomes (yacs), bacterial artificial chromosomes (bacs),phage, viral vectors, and the like.

Genomic RNA transcripts may be synthesized from the DNA template by anymethod known in the art. For example, the RNA can be synthesized fromthe DNA sequence in vitro using purified RNA polymerase in the presenceof ribonucleotide triphosphates and cap analogs in accordance withconventional techniques. Alternatively, the RNA may be synthesizedintracellularly after introduction of the DNA.

Further provided are cells containing the DNA sequences, genomic RNAtranscribed from the DNA sequences, and alphavirus vectors of theinvention. Exemplary cells include, but are not limited to, fibroblastcells, Vero cells, Baby Hamster Kidney (BHK) cells, Chinese HamsterOvary (CHO) cells, 293 cells, 293T cells, and chicken embryo fibroblastcells (e.g., DF-1 cells), macrophages, PBMC, monocytes, and dendriticcells.

The alphavirus DNA constructs, genomic RNA transcripts, and virusparticles produced therefrom are useful for the preparation ofpharmaceutical formulations, such as vaccines. In addition, the DNAclones, genomic RNA transcripts, and infectious viral particles of thepresent invention are useful for administration to animals for thepurpose of producing antibodies to the alphavirus, which antibodies maybe collected and used in known diagnostic techniques for the detectionof alphaviruses. Antibodies can also be generated to the viral proteinsexpressed from the DNAs disclosed herein. As another aspect of thepresent invention, the claimed DNA clones are useful as nucleotideprobes to detect the presence of alphavirus transcripts.

VII. Subjects, Pharmaceutical Formulations, Vaccines, and Modes ofAdministration.

The present invention finds use in both veterinary and medicalapplications. Suitable subjects include both avians and mammals, withmammals being preferred. The term “avian” as used herein includes, butis not limited to, chickens, ducks, geese, quail, turkeys and pheasants.The term “mammal” as used herein includes, but is not limited to,primates (e.g., simians and humans), bovines, ovines, caprines,porcines, equines, felines, canines, lagomorphs, rodents (e.g., rats andmice), etc. Human subjects include fetal, neonatal, infant, juvenile andadult subjects.

The invention may be used in either a therapeutic or prophylacticmanner. For example, in one embodiment, to protect against an infectiousdisease, subjects may be vaccinated prior to exposure, as neonates oradolescents. Adults that have not previously been exposed to the diseasemay also be vaccinated. In cancer patients, use of the present inventionmay be used in conjunction with other cancer therapies, e.g., before,during or after the surgical removal of tumors, chemotherapy orradiation.

In particular embodiments, the present invention provides apharmaceutical composition comprising an alphavirus vector of theinvention in a pharmaceutically-acceptable carrier or other medicinalagents, pharmaceutical agents, carriers, adjuvants, diluents, etc. Forinjection, the carrier will typically be a liquid. For other methods ofadministration, the carrier may be either solid or liquid, such assterile, pyrogen-free water or sterile pyrogen-free phosphate-bufferedsaline solution. For inhalation administration, the carrier will berespirable, and will preferably be in solid or liquid particulate form.As an injection medium, it is preferred to use water that contains theadditives usual for injection solutions, such as stabilizing agents,salts or saline, and/or buffers.

In other embodiments, the present invention provides a pharmaceuticalcomposition comprising a cell (e.g., a dendritic cell) that has beeninfected and genetically modified by an alphavirus vector in apharmaceutically-acceptable carrier or other medicinal agents,pharmaceutical agents, carriers, adjuvants, diluents, etc.

By “pharmaceutically acceptable” it is meant a material that is notbiologically or otherwise undesirable, e.g., the material may beadministered to a subject without causing any undesirable biologicaleffects. Thus, such a pharmaceutical composition may be used, forexample, in transfection of a cell ex vivo or in administering thealphavirus/antibody compositions or cells directly to a subject.

The cell to be administered the virus vectors can be of any type,including but not limited to neuronal cells (including cells of theperipheral and central nervous systems), retinal cells, epithelial cells(including dermal, gut, respiratory, bladder and breast tissueepithelium), muscle cells (including cardiac, smooth muscle, skeletalmuscle, and diaphragm muscle), pancreatic cells (including islet cells),hepatic cells (e.g., parenchyma), fibroblasts, endothelial cells, germcells, lung cells (including bronchial cells and alveolar cells),prostate cells, stem cells, progenitor cells, dendritic cells, and thelike. Alternatively, the cell is a cancer cell (including tumor cells).Moreover, the cells can be from any species of origin, as indicatedabove.

Alternatively, in embodiments of the invention, the cell is preferably acell is a bone marrow cell or a cell in the bone-associated connectivetissue. Other preferred cells, are cells of the periosteum, endosteumand tendons, generally within the epiphyses of the long bones adjacentto joints.

In still other embodiments, the cell is an antigen-presenting cell(e.g., a dendritic cell or a macrophage).

Cells that may be infected by the alphavirus vectors of the presentinvention further include, but are not limited to, polymorphonuclearcells, hemopoietic stem cells (including megakaryocyte colony formingunits (CFU-M), spleen colony forming units (CFU-S), erythroid colonyforming units (CFU-E), erythroid burst forming units (BFU-E), and colonyforming units in culture (CFU-C), erythrocytes, macrophages (includingreticular cells), monocytes, granulocytes, megakaryoctyes, lymphocytes,fibroblasts, osteoprogenitor cells, osteoblasts, osteoclasts, marrowstromal cells, chondrocytes and other cells of synovial joints.

The alphavirus vectors of the invention may be administered to elicit animmunogenic response (e.g., as an immunogenic composition or as avaccine for immunotherapy). Typically, immunological compositions of thepresent invention comprise an immunogenic amount of infectious virusparticles as disclosed herein in combination with apharmaceutically-acceptable carrier.

An “immunogenic amount” is an amount of the infectious virus particlesthat is sufficient to induce an immune response in the subject to whichthe pharmaceutical formulation is administered. Typically, a dosage ofabout 10³ to about 10¹⁵ infectious units, about 10⁴ to about 10¹⁰infectious units, about 10² to about 10⁶ infectious units; about 10³ toabout 10⁵ infectious units, about 10⁵ to about 10⁹ infectious units, orabout 10⁶ to about 10⁸ infectious units per dose is suitable, dependingupon the age and species of the subject being treated, and the immunogenagainst which the immune response is desired.

In other embodiments, a dosage of about 10³ to about 10⁴ infectiousunits, about 10⁴ to about 10⁵ infectious units, about 10⁴ to about 10⁶infectious units, about 10⁶ to about 10⁷ infectious units, about 10⁷ toabout 10⁸ infectious units, about 10⁶ to about 10⁷ infectious units,about 10⁹ to about 10¹⁰ infectious units, or about 10¹⁰ to about 10¹¹infectious units per dose is suitable.

In still other embodiments, the dosage is about 10³ to about 5×10³infectious units, about 5×10³ to about 10⁴ infectious units, about 10⁴to about 5×10⁴ infectious units, about 5×10⁴ to about 10⁵ infectiousunits, about 10⁵ to about 5×10⁵ infectious units, about 5×10⁵ to about10⁶ infectious units, about 10⁶ to about 5×10⁶ infectious units, about5×10⁶ to about 10⁷ infectious units, about 10⁷ to about 5×10⁷ infectiousunits, about 10⁷ to about 5×10⁷ infectious units, about 5×10⁷ to about10⁸ infectious units, about 10⁸ to about 5×10⁸ infectious units, orabout 5×10⁸ to about 10⁹ infectious units per dose.

In yet further embodiments, the dosage is about 10³, about 10⁴, about10⁵, about 10⁶, about 10⁷, about 10⁸, about 10⁹, or about 10¹⁰infectious units per dose.

Subjects and immunogens are as described above. In representativeembodiments, the alphavirus vector is an alphavirus replicon particle(e.g., a VEE replicon particle).

The terms “vaccination” or “immunization” are well-understood in theart. For example, the terms vaccination or immunization can beunderstood to be a process that increases a subject's immune reaction toantigen and therefore the ability to resist or overcome infection. Inthe case of the present invention, vaccination or immunization may alsoincrease the organism's immune response and resistance to invasion bycancer or tumor cells.

Any suitable vaccine and method of producing an immune response (i.e.,immunization) known in the art may be employed in carrying out thepresent invention, as long as an active immune response (preferably, aprotective immune response) against the antigen is elicited.

According to the present invention, administration of an alphavirusvector comprising one or more heterologous nucleotide sequences encodingan immunogen elicits an active immune response in the subject, and inparticular embodiments, the active immune response is a protectiveimmune response.

An “active immune response” or “active immunity” is characterized by“participation of host tissues and cells after an encounter with theimmunogen. It involves differentiation and proliferation ofimmunocompetent cells in lymphoreticular tissues, which lead tosynthesis of antibody or the development of cell-mediated reactivity, orboth.” Herbert B. Herscowitz, Immunophysiology: Cell Function andCellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, anactive immune response is mounted by the host after exposure toimmunogens by infection or by vaccination. Active immunity can becontrasted with passive immunity, which is acquired through the“transfer of preformed substances (antibody, transfer factor, thymicgraft, interleukin-2) from an actively immunized host to a non-immunehost.” Id.

A “protective” immune response or “protective” immunity as used hereinindicates that the immune response confers some benefit to the subjectin that it prevents or reduces the incidence of disease. Alternatively,a protective immune response or protective immunity may be useful in thetreatment of disease, in particular cancer or tumors (e.g., by causingregression of a cancer or tumor and/or by preventing metastasis and/orby preventing growth of metastatic nodules). The protective effects maybe complete or partial, as long as the benefits of the treatmentoutweigh any disadvantages thereof.

Vaccination can be by any means known in the art, but is preferably byoral, rectal, transmucosal, intranasal, topical, transdermal,inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal,intramuscular, intraperitoneal and intraarticular) administration, andthe like. Alternatively, the alphavirus vector may be directlyadministered by implant or injection into or near a tumor. In the caseof animal subject, injection may be into the footpad.

In particular embodiments of the invention, administration is bysubcutaneous or intradermal administration. Subcutaneous and intradermaladministration may be by any method known in the art, including but notlimited to injection, gene gun, powderject device, bioject device,microenhancer array, microneedles, and scarification (i.e., abrading thesurface and then applying a solution comprising the virus).

In other embodiments, administration is to the limb of the subject,e.g., by subcutaneous or intradermal administration. In still otherparticular embodiments, administration to the limb (e.g., bysubcutaneous or intradermal routes) is to the front limb of the subject,i.e., in the case of bipeds such as a primate, administration is to thearm of the subject and in the case of a quadruped, administration is tothe front leg. In still further embodiments, administration is to thelower part of the arm (e.g., in a primate, below the elbow).

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Alternatively,one may administer these reagents as an aerosol, or in a local ratherthan systemic manner, for example, in a depot or sustained-releaseformulation.

In other preferred embodiments, the alphavirus vector is administeredintramuscularly, more preferably by intramuscular injection or by localadministration (as defined above).

In other preferred embodiments, the alphavirus vectors of the presentinvention are administered to the lungs. The alphavirus vectorsdisclosed herein may be administered to the lungs of a subject by anysuitable means, but are preferably administered by administering anaerosol suspension of respirable particles comprised of the alphavirusvectors, which the subject inhales. The respirable particles may beliquid or solid. Aerosols of liquid particles comprising the alphavirusvectors may be produced by any suitable means, such as with apressure-driven aerosol nebulizer or an ultrasonic nebulizer, as isknown to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729.Aerosols of solid particles comprising the virus vectors may likewise beproduced with any solid particulate medicament aerosol generator, bytechniques known in the pharmaceutical art.

The present invention further provides a method of delivering a nucleicacid to a cell (e.g., to produce an immune response or for therapy). Forin vitro methods, the virus may be administered to the cell by standardviral transduction methods, as are known in the art. Cells to beadministered the alphavirus vector are as described above. Preferably,the virus particles are added to the cells at the appropriatemultiplicity of infection according to standard transduction methodsappropriate for the particular target cells. Titers of virus toadminister can vary, depending upon the target cell type and theparticular virus vector, and may be determined by those of skill in theart without undue experimentation.

In particular embodiments of the invention, cells are removed from asubject, the alphavirus vector is introduced therein, and the cells arethen replaced back into the subject. Methods of removing cells fromsubject for treatment ex vivo, followed by introduction back (e.g.,intravenously) into the subject are known in the art. Alternatively, thealphavirus vector is introduced into cells from another subject, intocultured cells, or into cells from any other suitable source, and thecells are administered to a subject in need thereof. Preferably, if thesubject's own cells are not used, the cells are HLA compatible with thesubject's HLA type. The modified cell may be administered according to amethod of ex vivo gene therapy or to provide immunity to a subject(e.g., by introducing a nucleotide sequence encoding an immunogen intoan antigen producing cell, such as a dendritic cell).

Dosages of the cells to administer to a subject will vary upon the age,condition and species of the subject, the type of cell, the nucleic acidbeing expressed by the cell, the mode of administration, and the like.Typically, at least about 10² to about 10⁸, preferably about 10³ toabout 10⁶ cells, will be administered per dose. Preferably, the cellswill be administered in a “immunogenic amount” (as describedhereinabove) or a “therapeutically-effective amount”.

Particular embodiments of the present invention are described in greaterdetail in the following non-limiting examples.

EXAMPLE 1 Materials and Methods

Virus: VEE replicon particles (VRP) expressing either influenza virushemagglutinin (HA-VRP-3000, HA-VRP-3014, and HA-VRP-3042), greenfluorescent protein (GFP-VRP-3000, GFP-VRP-3014, and GFP-VRP-3042), orHIV Clade C gag (HIV_(gag)-VRP-3000, HIV_(gag)-VRP-V3014, andHIV_(gag)-VRP-3042) were prepared as previously described (MacDonald andJohnston, 2000 J. Virology 74:914, Pushko et al. 1997 Virology 239:389).Briefly, RNA transcripts from replicon cDNA plasmids encoding theappropriate heterologous gene were co-electroporated with RNAtranscripts from two helper constructs encoding either VEE capsid or VEEglycoprotein genes into baby hamster kidney (BHK) cells. VRP wereharvested directly from the culture supernates 24 hr followingelectroporation and titered on BHK cells. For these studies, VRP wereproduced using a glycoprotein helper that contained the V3014attenuating mutations, i.e., an Ala→Thr mutation at E1 position 272, aGlu→Lys mutation at E2 position 209, and a Ile→Asn mutation at E2position 239 (Davis et al., (1991) Virology 183:20), V3040 attenuatingmutation at E1 253 (Phe→Ser) or the V3042 attenuating mutation at E1 81(Phe→Ile).

Mice and Cells: Seven- to eight-week-old female CD1 out bred mice(Charles River Laboratory) were inoculated subcutaneously (sc) in theleft rear foot pad with 5×10⁵ infectious units (IU) of VEE viralreplicon particles (VRP) unless otherwise specified. Mice were perfusedwith 4% paraformaldehyde (PFA) in PBS 24 hr post-inoculation (pi) andthe draining popliteal lymph nodes were removed to PFA. Fixed frozensections were analyzed by fluorescent microscopy for cells expressingGFP.

Bone marrow (BM) cells were isolated from the femurs of C57BL6 mice.Cells were grown as previously described. Briefly, marrow was flushedfrom femurs and tibia and resuspended in PBS. Cells were washed andre-suspended in RPMI1640 supplemented with 10% FBS, L-glutamine,nonessential amino acids, sodium pyruvate, 50 μM β-2-mercaptoethanol,and 25 mM HEPES. Cultures were supplemented with 0.1 ng/ml GM-CSF aloneor with either 5% conditioned culture medium from the epidermalfibroblast cell line, NS46 (Xu et al., (1995) J. Immunol. 154:2697) or 1ng/ml IL4 and grown on standard tissue culture plates.

VEE Replicon Particles (VRP) Inoculation of Macaques: VEE repliconparticles packaged using wild-type glycoprotein coats were inoculatedinto rhesus macaques in each leg (5 cm lateral to the inguinal triangle)with 1×10⁴ or 1×10⁷ IU VRP-GFP or VRP-HA in 0.5 ml PBS. Inguinal lymphnodes were harvested 18 hours post inoculation, fixed immediately inparaformaldehyde, and processed for microscopy.

ELISA: Antibody assays were performed as described in Davis et al.(1996) J. Virol. 70:3781-3787. Gradient-purified PR/8/34 influenza viruswas used as an antigen and horseradish peroxidase (HRP)-conjugatedanti-mouse immunoglobulin G (IgG) or HRP-conjugated goat anti-mouse IgAwas used as the second antibody.

In Situ Hybridization Analysis: Tissues were prepared as described byCharles et al. (1995) Virology 208:662-671 and Grieder et al. (1995)Virology 206:994-1006. Assays were performed as described in Davis etal. (1996) J. Virol. 70:3781-3787.

EXAMPLE 2 Anti-HA Response to VRP Immunization

The effect of dosage on the primary and secondary response in HAvector-immunized mice was examined. VRP-replicons were administered at0.1 to 10,000 IU. Four weeks post-inoculation, the mice were bled andELISA assays for anti-HA response at varying doses of HA-VRP-3000(wild-type) and HA-VRP-3014 (attenuated) were performed. The results aredepicted in FIG. 1. In the same animals at four weeks, a secondinoculation of VRP was administered. Four weeks after the secondinoculation, ELISA assays for secondary Anti-HA response were performedand are shown in FIG. 2. These results indicate that mutations in thecoat protein have a significant effect on the HA replicon induced immuneresponse. At or below a dose of 10 IU per mouse, little primary orsecondary response from immunization with HA-VRP-3014 (mutant coatprotein) was observed in comparison to HA-VRP-3000 (wild-type). As thevector dosage is increased (100-10,000 IU), response to HA-VRP-3014 asdetermined from ELISA titer improves in both primary and secondaryresponses. The secondary response to HA-VRP-3014 at a dose of 10,000 IUapproached that of the wild-type (HA-VRP-3000).

EXAMPLE 3 HIV Clade C Gag-Specific CTL Response in Mice

CTL response to HIV Clade gag in mice primed and boosted with 100 IU ofHIV_(gag)-VRP-3000 is depicted in FIG. 3. Groups of six mice were primedand boosted four weeks after initial inoculation. HIV_(gag)-specific CTLresponses were determined according to a standard chromium release assay(Hioe and Frelinger (1995) Mol. Immunol. 32:725-731) one week followingthe boost at various effector to target (E:T) cell ratios. A Class 1 H-2K^(d) restricted Gag peptide (AMQMLKETI) was used as the relevantpeptide. An irrelevant H-2K^(d) restricted HA (influenza virushemagglutinin) peptide was used as a negative control. The percentspecific lysis was calculated as:[(experimental release−spontaneous release)/(maximum release−spontaneousrelease)]×100.Spontaneous release was defined as counts per minute released fromtarget cells in the absence of effector cells, and maximum release wasdefined as counts per minute released from target cells lysed with 2.5%Triton X-100. HIV_(gag)-specific CTL activity was defined as 10% lysisabove controls. The results shown in FIG. 3 indicate that HIV_(gag)-VRPreplicons can induce a HIV_(gag)-specific CTL response. The CTL responseto HIV Clade gag in mice primed and boosted with HIV_(gag)-VRP repliconspackaged in different coat proteins (wild-type HIV_(gag)-VRP-3000 andmutant HIV_(gag)-VRP-3014) at varying doses is depicted in FIG. 4. Theseresults indicate that the replicon coat protein has an effect on theobserved CTL response in primed and boosted mice. VRP-3014 (mutant coatproteins) elicits a weaker CTL response than VRP-3000 (wild-type).

EXAMPLE 4 Envelope Effect on HA Response in Mice

The effect of envelope coat protein on HA replicon inducedimmunogenicity is shown in FIG. 5. ELISA titers comparing HA response toHA replicons with different envelopes indicate that mutations in thecoat protein do not necessarily have deleterious effects on antibodyresponse. The E1 81 mutation HA3042 elicits a greater HA response thaneven the wild-type HA3000, while HA3014 elicits weaker responses thanthe wild-type. HA3040 exhibits only a modest depression as compared withthe wild-type. These results suggest that the attenuated coat viruses,3040 and 3042, are safe without substantially adverse effects onefficacy.

EXAMPLE 5 Effect of Route of Administration on HA Response in Mice

HA replicons were introduced by subcutaneous inoculation in the back ofthe neck, and by intradermal inoculation in the rear thigh. Four weeksfollowing the first inoculation with 10³ IU VRP, a second 10³ IU dose ofVRP was administered, and the mice were bled four weeks thereafter. TheELISA antibody titers are shown in FIG. 6. The results indicate thatintradermal inoculation of HA-VRP generally elicits a stronger secondaryresponse than subcutaneous inoculation. HA3042 produced a strongresponse by all routes of administration. In contrast, wild type, HA3014and HA3040 gave a stronger response with intradermal administration ascompared with subcutaneous administration. Wild type and attenuatedviruses elicited a strong response with inoculation via the footpad. Inall cases, HA3042 elicits the strongest ELISA response. The differencein response is most apparent in subcutaneous inoculations, with lesserdifferences observed for intradermal and footpad inoculations.

EXAMPLE 6 Dosage and Route Effect on Dendritic Cell Infection inMacaques

GFP-VRP-3000 is administered to four rhesus macaques by eithersubcutaneous or intradermal inoculation, 5 cm lateral to the inguinaltriangle. Two animals receive a high dose (10⁷ IU of VRP), and twoanimals receive a low dose (10⁴ IU of VRP) of vector. The right leg ofeach animal receives a subcutaneous inoculation of vector, while theleft leg receives an intradermal inoculation of vector. Eighteen hourspost-inoculation, simple excision of the inguinal lymph nodes isperformed and processed for fluorescence microscopy. The results fromthe fluorescence microscopy performed on these tissues indicates theeffect of the route (subcutaneous vs. intradermal) and dosage ondendritic cell infection.

EXAMPLE 7 Quantitation of Immune Response to Vaccination in Macaques

HA-VRP-3000 is administered at 10⁵ IU in 0.5 ml PBS to two groups offour animals and boosted at 1 month. One group of animals receives thevaccine via subcutaneous inoculation, the other group receives thevaccine via intradermal inoculation. Inoculations are performed asoutlined in Example 6. Blood is drawn for antibody determinations(anti-HA) at 0, 1, 2, and 4 months by ELISA. The results from this studyallow the direct quantification of the immune response resulting fromthe different routes of vaccine administration.

EXAMPLE 8 Effect of Coat Protein on Dendritic Cell Infection in Macaques

GFP-VRP-3000 (wild-type coat protein), along with GFP-VRP-3014 andGFP-VRP-3042 (mutant coat proteins) are used in this study. The study isdivided into two groups: high dose (10⁷ IU), and low dose (10⁴ IU). Eachanimal receives one dose of vaccine (in 0.5 ml PBS) in each leg (5 cmlateral from the inguinal triangle) via the most effective route ofadministration as determined in Example 6. Each animal (twelve total)are vaccinated in the following scheme:

Simple excision of inguinal lymph nodes is performed from both sidesusing sterile technique and standard surgical methods 18 hourspost-inoculation. The nodes are immediately be fixed in paraformaldehydeand processed for microscopy. The results examine the effect of dose andVRP coat protein on dendritic cell targeting of VRP infection.

EXAMPLE 9 Effect of Coat Protein on Immune Response in Macaques

HA-VRP-3000 (wild-type coat protein), along with HA-VRP-3014 andHA-VRP-3042 (mutant coat proteins) are used in this study. Three groupsof four animals are used in this study, the first group is inoculatedwith HA-VRP-3000, the second group is inoculated with HA-VRP-3014, andthe third group is inoculated with HA-VRP-3042. Each animal isinoculated with 10⁵ IU in 0.5 ml PBS of the appropriate vector at 0 and1 month via the most effective route as determined according to Example6. Animals are bled at 0, 1, 2, and 4 months for Anti-HA response. Theresults correlate the effect of VRP coat protein on immune responseelicited by the vaccine.

EXAMPLE 10 Dendritic Cell Infection in Draining Lymph Nodes of Macaques

GFP-VRP-3000 (10⁴ IU VRP in 0.5 ml PBS) was administered to four rhesusmacaques, 5 cm lateral to the inguinal triangle as described in Example6. Eighteen hours post-inoculation, simple excision of the inguinallymph nodes were performed and processed for fluorescence microscopy asdescribed in Example 1 (FIG. 7). The positive fluorescence observedindicates that dendritic cells are targeted by wild-type GFP-VRP inmacaques.

EXAMPLE 11 Heparin Affinity Chromatography of Mutagenized ViralParticles

Heparin affinity chromatography can be performed using any of severalcommercially available resins to which heparin has been bound. Thesource of heparin in these columns is variable; current commerciallyavailable resins use porcine heparin, but other sources can be usedeffectively.

A. Pharmacia HiTrap® Heparin

Columns of Pharmacia HiTrap® Heparin (cat no. 17-0407-01, AmershamPharmacia Biotech) are pre-equilibrated with 25 mM HEPES/0.25 M NaCl, pH7.5, and then loaded with mutagenized virus preparations as describedabove. Non or weakly binding mutants are collected in the first eluantsfrom the column, i.e. where the non-bound materials elute.

B. Heparin Sepharose 6 Fast Flow® resin

Heparin Sepharose 6 Fast Flow® resin (catalog no. 90-1000-2; AmershamPharmacia Biotech) is supplied as a bulk resin which allows various sizecolumns to be packed as needed. A 6 ml column is prepared by packing theHeparin Sepharose 6 Fast Flow® bulk resin in a BioRad® Econo-Columnchromatography column, then pre-equilibrated with 25 mM HEPES/0.12 MNaCl, pH 7.5. Mutagenized viral preparations are loaded onto the column,and non- or weakly binding mutants are collected in the first eluantsfrom the column.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A composition comprising a population of infectious, attenuated,alphavirus replicon particles in an immunogenically effective dosage,wherein each of said alphavirus particles comprises: (a) a virion shellcomprising Venezuelan Equine Encephalitis (VEE) structural proteins,wherein said virion shell further comprises an attenuating mutation inthe E1 glycoprotein; (b) a recombinant alphavirus replicon RNAcomprising a heterologous nucleotide sequence encoding an immunogen,wherein said heterologous nucleotide sequence is operably associatedwith a promoter, wherein said immunogenically effective dosage comprisesa number of infectious alphavirus particles that is (i) substantiallythe same as or substantially less than the immunogenically effectivedosage of a comparable alphavirus having a wild-type VEE virion shell or(ii) is less than about 1 00-fold more than the immunogenicallyeffective dosage of a comparable alphavirus having a wild-type VEEvirion shell.
 2. The composition of claim 1, wherein saidimmunogenically effective dosage comprises substantially the same numberof infectious alphavirus particles as an immunogenically effectivedosage of a comparable virus having a wild-type VEE virion shell.
 3. Thecomposition of claim 1, wherein said immunogenically effective dosagecomprises a substantially lower number of infectious alphavirusparticles than an immunogenically effective dosage of a comparablealphavirus having a wild-type VEE virion shell.
 4. A compositioncomprising a population of infectious, attenuated, alphavirus repliconparticles in an immunogenically effective dosage, wherein each of saidalphavirus particles comprises: (a) a virion shell comprising VenezuelanEquine Encephalitis (VEE) structural proteins, wherein said virion shellfurther comprises an attenuating mutation in the E1 glycoprotein; (b) arecombinant alphavirus replicon RNA comprising a heterologous nucleotidesequence encoding an immunogen, wherein said alphavirus particlesexhibit only weak or no detectable binding to heparin.
 5. Thecomposition of claim 1, wherein said attenuating mutation in the E1glycoprotein comprises an attenuating mutation in the fusogenic peptideregion.
 6. The composition of claim 1, wherein said attenuating mutationin the E1 glycoprotein comprises an attenuating mutation selected fromthe group consisting of (i) an attenuating mutation at E1 glycoproteinamino acid position 81, and (ii) an attenuating mutation at E1glycoprotein amino acid position
 253. 7. The composition of claim 6,wherein said VEE virion shell comprises a Phe→Ile attenuating mutationat E1 glycoprotein amino acid position
 81. 8. The composition of claim6, wherein said VEE virion shell comprises a Phe→Ser attenuatingmutation at E1 glycoprotein amino acid position
 253. 9. The compositionof claim 1, wherein said composition comprises about 10² to about 10⁶infectious alphavirus particles.
 10. The composition of claim 1, whereinsaid composition comprises about 10³ to about 10⁵ infectious alphavirusparticles.
 11. The composition of claim 1, wherein said compositioncomprises about 10⁵ to about 10⁹ infectious alphavirus particles. 12.The composition of claim 11, wherein said composition comprises about10⁶ to about 10⁸ infectious alphavirus particles.
 13. The composition ofclaim 1, wherein said recombinant alphavirus replicon RNA is arecombinant VEE replicon RNA.
 14. The composition of claim 1, whereinsaid promoter is an alphavirus 26S subgenomic promoter.
 15. Thecomposition of claim 1, wherein said immunogen is a cancer immunogen.16. The composition of claim 1, wherein said immunogen is an infectiousdisease immunogen.
 17. The composition of claim 16, wherein saidimmunogen is selected from the group consisting of a bacterialimmunogen, a viral immunogen, and a protozoa immunogen.
 18. Thecomposition of claim 1, wherein said immunogen is a SimianImmunodeficiency Virus (SIV) immunogen or a Human Immunodeficiency Virus(HIV) immunogen.
 19. The composition of claim 18, wherein said immunogenis a SIV or HIV immunogen selected from the group consisting of a gag,env, ref, tat, nef and pol gene product, and a combination thereof. 20.The composition of claim 1, wherein said replicon RNA lacks sequencesencoding the VEE structural proteins.
 21. A pharmaceutical formulationcomprising the composition of claim 1 in a pharmaceutically acceptablecarrier.
 22. A method of producing an immune response in a subject,comprising administering to the subject an immunogenically effectiveamount of a composition according to claim
 1. 23. A method of producingan immune response in a subject, comprising: (a) administering ex vivoto a plurality of cells a composition according to claim 1, and (b)administering an immunogenically effective amount of the cells to thesubject.
 24. The method of claim 23, wherein the plurality of cellscomprises dendritic cells.
 25. The method of claim 22, wherein aprotective immune response is induced in the subject.
 26. The method ofclaim 22, wherein said administering step is carried out by subcutaneousadministration.
 27. The method of claim 22, wherein said administeringstep is carried out by intradermal administration.
 28. The method ofclaim 22, wherein said administering step is carried out byadministration to a limb of the subject.
 29. The method of claim 28,wherein said administering step is to a front limb of the subject. 30.The method of claim 22, wherein the subject is a mammalian subject. 31.The method of claim 30, wherein the subject is selected from the groupconsisting of a primate subject, a pig, a cow, a dog and a cat.
 32. Themethod of claim 31, wherein the subject is a human subject.
 33. Themethod of claim 32, wherein the subject has, or is at risk ofdeveloping, AIDS.
 34. The method of claim 24, wherein the heterologousnucleotide sequence is introduced into the dendritic cells and thedendritic cells express the immunogen.
 35. The method of claim 24,wherein said contacting step is carried out in vitro.
 36. The methodaccording to claim 24, wherein said contacting step is carried out invivo.